The neurotrophins are a family of structurally and functionally related proteins, including Nerve Growth Factor (NGF), Brain-Derived Neurotrophic Factor (BDNF), Neurotrophin-3 (NT-3), Neurotrophin-4/5 (NT-4/5) and Neurotrophin-6 (NT-6). These proteins promote the survival and differentiation of diverse neuronal populations in both the peripheral and central nervous systems (Hefti, 1986; Hefti and Weiner, 1986; Levi-Montalcini, 1987; Barde, 1989; Leibrock et al., 1989; Maisonpierre et al., 1990; Rosenthal et al., 1990; Hohn et al., 1990; Gotz et al., 1994; Maness et al., 1994) and are involved in the pathogenesis of diverse neurological disorders. Neurotrophins exert many of their biological effects through specific interactions with a class of transmembrane receptor tyrosine kinases (trkA, trkB and trkC) (Kaplan et al., 1991; Klein et al., 1991, 1992; Soppet et al., 1991; Squinto et al., 1991; Berkemeier et al., 1991; Escandon et al., 1993; Lamballe et al., 1991). Specificity of neurotrophin action results from their selective interactions with the trk receptors. That is, trkA only binds NGF (Kaplan et al., 1991; Klein et al., 1991); trkB binds BDNF and NT-4/5 (Soppet et al., 1991; Squinto et al., 1991; Berkemeier et al., 1991; Escandon et al., 1993; Lamballe et al., 1991; Klein et al., 1992; Vale and Shooter, 1985; Barbacid, 1993); and trkC exclusively binds NT-3 (Lamballe et al., 1991; Vale and Shooter, 1985). This is particularly evident when the trk receptors are coexpressed with the common neurotrophin receptor p75NTR. (For review see Meakin and Shooter, 1992; Barbacid, 1993; Chao, 1994; Bradshaw et al., 1994; Ibxc3xa1xc3x1ez, 1995).
The common neurotrophin receptor p75NTR is a transmembrane glycoprotein structurally related to the tumor necrosis factor and CD-40 receptors (Meakin and Shooter, 1992; Rydxc3xa9n and Ibxc3xa1xc3x1ez, 1996). As all neurotrophins bind to p75NTR with similar affinity (Rodriguez-Txc3xa9bar et al., 1990; Hallbxc3x6xc3x6k et al., 1991; Rodriguez-Txc3xa9bar et al., 1992; Ibxc3xa1xc3x1ez, 1995), neurotrophin specificity is conventionally thought to be caused by the binding selectivity for trk receptors which are differentially expressed in different neuronal populations (Ibxc3xa1xc3x1ez, 1995). However, accumulated experimental data on neurotrophin activity reveal important functional aspects of p75NTR (Heldin et al., 1989; Jing et al., 1992; Herrmann, 1993; Barker and Shooter, 1994; Dobrowsky et al., 1994, Matsumoto et al., 1995; Marchetti et al., 1996; Washiyama et al., 1996). The common neurotrophin receptor enhances functions and increases binding specificity of trk receptors (Barker and Shooter, 1994; Mahadeo et al., 1994; Chao and Hempstead, 1995; Rydxc3xa9n and Ibxc3xa1xc3x1ez, 1996). In addition, p75NTR possesses unique, trk-independent signaling properties which involve ceramide production through activation of the sphingomyelin cycle (Dobrowsky et al., 1994), apoptosis (cell death) (Van der Zee et al., 1996; Cassacia-Bonnefil et al., 1996; Frade et al., 1996), and activation of the transcription factor NFxcexaB (Carter et al., 1996). Recently, p75NTR has been demonstrated to participate in human melanoma progression (Herrmann et al., 1993; Marchetti et al., 1996). Furthermore, NGF and NT-3 increase the production of heparin by 70W melanoma cells, which is associated with their metastatic potential (Marchetti et al., 1996). Although this effect has been shown to be mediated by the common neurotrophin receptor, neither BDNF nor NT-4/5 appeared to be active.
Due to the implication of NGF/p75NTR binding in various disease states, a need exists for pharmaceutical agents and methods of use thereof for interfering with the binding of NGF to the p75NTR common neurotrophin receptor.
The present invention relates to the discovery of molecular structural features which contribute to the ability of a compound to inhibit the binding of NGF to the common neurotrophin receptor p75NTR. Compounds which have these features are of use, for example, for inhibiting binding of NGF to p75NTR. Such compounds can also be used to treat a patient having a condition which is mediated, at least in part, by the binding of NGF to p75NTR.
In one embodiment, the present invention relates to compositions which inhibit the binding of nerve growth factor to the p75NTR common neurotrophin receptor and methods of use thereof.
In one embodiment, the compound which inhibits binding of nerve growth factor to p75NTR comprises at least two of the following: (1) a first electronegative atom or functional group positioned to interact with Lys34 of nerve growth factor; (2) a second electronegative atom or functional group positioned to interact with Lys95 of nerve growth factor; (3) a third electronegative atom or functional group positioned to interact with LyS88 of nerve growth factor; (4) a fourth electronegative atom or functional group positioned to interact with Lys32 of nerve growth factor; and (5) a hydrophobic moiety which interacts with the hydrophobic region formed by amino acid residues of nerve growth factor, including Ile31, Phe101 and Phe86. Such inhibitors, preferably, bind nerve growth factor via at least two of the foregoing interactions.
In one embodiment, compounds which inhibit binding of nerve growth factor to p75NTR have Formula 1, 
In Formula 1, D1, D2, E1, E2 and G are each, independently, an sp2-hybridized carbon or nitrogen atom. One of X1 and X2 is a hydrogen atom or absent, while the other is an electronegative atom or an electronegative functional group. R and R2 are each, independently, an electronegative atom or an electronegative functional group, such as O, S, CH2, or NR3, where R3 is H, alkyl, preferably C1-C6-alkyl, or aryl, such as phenyl. R, R2 and one of X1 and X2 can also each be, independently, an electronegative atom or functional group, such as alkylcarbonyl; alkylthiocarbonyl; alkoxycarbonyl; aminocarbonyl; xe2x80x94OH; xe2x80x94CN; xe2x80x94CO2H; xe2x80x94SO3H; xe2x80x94SO2H; xe2x80x94PO3H2; xe2x80x94NO2; xe2x80x94ONO2, xe2x80x94CNO, xe2x80x94SH, xe2x80x94CNS, xe2x80x94OSO3H, xe2x80x94OC(O)(OH); halomethyl, dihalomethyl or trihalomethyl group or a fluorine, chlorine, bromine or iodine atom. Y is N, O, S, Cxe2x80x94L or Nxe2x80x94L, where L is H, alkyl, preferably C1-C6-alkyl, or an electronegative atom or functional group, such as, but not limited to, alkylcarbonyl; alkylthiocarbonyl; alkoxycarbonyl; aminocarbonyl; xe2x80x94OH; xe2x80x94CN; xe2x80x94CO2H; xe2x80x94SO3H; xe2x80x94SO2H; xe2x80x94PO3H2; xe2x80x94NO2; xe2x80x94ONO2,xe2x80x94CNO, xe2x80x94SH, xe2x80x94CNS, xe2x80x94OSO3H, xe2x80x94OC(O)(OH); halomethyl, dihalomethyl or trihalomethyl groups or a halogen atom, such as a fluorine, chlorine, bromine or iodine atom. Z and Z1 are each, independently, O, S, CH, C(O), N, NH, N-alkyl, N-cycloalkyl and Nxe2x80x94P, where P is a carbohydrate moiety, such as a monosaccharide group, for example, a fucosyl, glucosyl, galactosyl, mannosyl, fructosyl, gulosyl, idosyl, talosyl, allosyl, altrosyl, ribosyl, arabinosyl, xylosyl or lyxosyl group. T1 and T2 are each, independently, an sp2- or sp3-hybridized carbon or nitrogen atom. a, b, and c are each 0 or 1, provided that at least one of b and c is 1. R1 is a monocyclic or polycyclic aryl or heteroaryl, monosaccharide or oligosaccharide, alkyl, cycloalkyl, arylalkyl, alkylamino or alkoxy group which is substituted with at least one substituent selected from the group consisting of electronegative atoms and electronegative functional groups.
It will be appreciated that in this and the following structures, the lines connecting the variables can be single or double bonds. In addition, hydrogen atoms are added to the variables as necessary to complete the valence of the atom.
In another embodiment, compounds have Formula 3, In another embodiment, the NGF/p75NTR binding inhibitor has Formula 3 
where D1, D2, X1, X2, Y, E1, E2, T1, T2, R, G, R1, R2, and c have the meanings given above for these variables in Formula 1. Y1, Y2, and Y3 are independently selected from the identities given for Y in Formula 1. E3 and E4 are each, independently, an sp2-hybridized carbon or nitrogen atom, and d and h are, independently, 0 or 1.
In another embodiment, compounds which inhibit the binding of nerve growth factor to p75NTR have Formula 2, 
In Formula 2, D1, D2, E1, E2, E3, E4 and G are each, independently, an sp2-hybridized carbon or nitrogen atom. One of X1 and X2 is a hydrogen atom or absent, while the other is an electronegative atom or an electronegative functional group. R, R2 and R4 are each, independently, an electronegative atom or electronegative functional group, such as O, S, CH2, or NR3, where R3 is H, OH, alkyl, preferably C1-C6-alkyl, or aryl, such as phenyl. R, R2 and one of X1 and X2 can also each be, independently, an electronegative atom or functional group, such as alkylcarbonyl; alkylthiocarbonyl; alkoxycarbonyl; aminocarbonyl; xe2x80x94OH; xe2x80x94CN; xe2x80x94CO2H; xe2x80x94SO3H; xe2x80x94SO2H; xe2x80x94PO3H2; xe2x80x94NO2; xe2x80x94ONO2, xe2x80x94CNO, xe2x80x94SH, xe2x80x94CNS, xe2x80x94OSO3H, xe2x80x94OC(O)(OH); halomethyl, dihalomethyl or trihalomethyl group or a fluorine, chlorine, bromine or iodine atom. Y is N, O, S, Cxe2x80x94L or Nxe2x80x94L, where L is H, alkyl, preferably C1-C6-alkyl, or an electronegative atom or functional group, such as, but not limited to, alkylcarbonyl; alkylthiocarbonyl; alkoxycarbonyl; aminocarbonyl; xe2x80x94OH; xe2x80x94CN; xe2x80x94CO2H; xe2x80x94SO3H; xe2x80x94SO2H; xe2x80x94PO3H2; xe2x80x94NO2; xe2x80x94ONO2, xe2x80x94CNO, xe2x80x94SH, xe2x80x94CNS, xe2x80x94OSO3H, xe2x80x94OC(O)(OH); halomethyl, dihalomethyl or trihalomethyl groups or a halogen atom, such as a fluorine, chlorine, bromine or iodine atom. Z and Z1 are each, independently, O, S, CH, C(O), N, NH, N-alkyl, N-cycloalkyl and Nxe2x80x94P, where P is a carbohydrate moiety, such as a monosaccharide group, for example, a fucosyl, glucosyl, galactosyl, mannosyl, fructosyl, gulosyl, idosyl, talosyl, allosyl, altrosyl, ribosyl, arabinosyl, xylosyl or lyxosyl group. T1, T2 and T3 are each, independently, an sp2- or sp3-hybridized carbon or nitrogen atom. When f is 0, T3 can further have the meanings given for Z and Z1 above. a, b, c, d, e, f, g, h and i are each 0 or 1, provided that at least one of b and c is 1, at least one of d and e is 1 and at least one of f and i is 1. R1 is a monocyclic or polycyclic aryl or heteroaryl, monosaccharide or oligosaccharide, alkyl, cycloalkyl, arylalkyl, alkylamine or alkoxy group which is substituted with at least one substituent selected from the group consisting of electronegative atoms and electronegative functional groups.
In another embodiment, a compound which inhibits the binding of NGF to p75NTR has Formula 5, 
wherein D1, D2, X1, X2, E1, E2, E3, T1, T2, T3, Z, G, R, R1, R2, R4, b, e, f, i, and c have the meanings given for these variables in Formula 2. Y1, Y2, and Y3 are independently selected from the identities given for Y in Formula 2, and h is 0 or 1. E5 and E6 are each, independently, an sp2-hybridized carbon or nitrogen atom, and g is 0 or 1. Ring 4 can be further unsubstituted or substituted with one or more substituents, such as alkyl or aryl groups.
In another embodiment, the invention provides a pharmaceutical composition comprising at least one compound of the invention, or pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable carrier or excipient.
The invention also provides a method of inhibiting the binding of nerve growth factor to the p75NTR receptor. The method comprises contacting cells which express the p75NTR receptor with a nerve growth factor/p75NTR binding inhibitor of the invention in an amount which is sufficient to inhibit binding of nerve growth factor to the p75NTR receptor. The method can be practiced in vivo or in vitro.
In another embodiment, the invention relates to a method of treating a condition in a patient which is mediated by the binding of nerve growth factor to the p75NTR receptor. The method comprises administering to the patient a therapeutically effective amount of a nerve growth factor/p75NTR binding inhibitor of the invention. Preferably, the compound to be administered selectively inhibits the binding of nerve growth factor to p75NTR in cells which do not express the NGF receptor trkA.